Kit, a Device and a Method for Detecting Copy Number of Fetal Chromosomes or Tumor Cell Chromosomes

ABSTRACT

The invention relates to a kit, a device and a method for detecting the copy number of fetal chromosomes and tumor cell chromosomes. The method for detecting the copy number of fetal chromosomes or tumor cell chromosomes of the invention includes the following steps: collecting maternal plasma or plasma of tumor patient; separating the plasma from blood cells in blood; preparing Deoxyribonucleic Acids (DNA) in the plasma into a sequencing library; sequencing the DNA sequencing library; comparing a sequencing result with a genomic sequence map to determine which chromosome the DNA sequence comes from and the length of each DNA sequence; and calculating the ratio of the DNA segments from the chromosomes to be detected to all DNA segments in the same sample by a sequencing and comparison result of DNA, correcting the ratio according to a GC content of the DNA segments from the chromosomes to be detected, and calculating the variation of the corrected ratio of the DNA segments from the chromosomes to be detected in a sample to be detected, and determining the copy number of the chromosomes to be detected according to degree of variation.

FIELD OF THE INVENTION

The invention relates to a kit, a device and a method for detecting thecopy number of fetal chromosomes or tumor cell chromosomes.

BACKGROUND OF THE INVENTION

The copy number abnormality of chromosomes is closely linked with humandiseases. The chromosome abnormality occurs in fetal cells carried withgenetic disease and tumor cells. On average, 9 of 1000 newborns maycarry diseases caused by copy number abnormality of chromosomes (1).Therefore, it is important to detect the copy number of chromosomesbefore children have not been born yet. However, currently useddiagnosis methods including amniocentesis and ovine chorionic belong toinvasive methods, which bring certain risks to pregnant women andfetuses. Serum protein markers and ultrasonic waves are used fordetecting whether the fetuses suffer diseases due to copy numberabnormality of chromosomes, although it is noninvasive, pathogenicfactors are not detected directly, so the accuracy and sensitivity isnot good (2). There is also a problem that the diseases due to the copynumber abnormality of chromosomes cannot be found as soon as possible.This situation prompts researchers to develop an accurate and highlysensitive noninvasive diagnosis and detection method.

Since fetal DNA in maternal blood have been found (3), diagnosing anddetecting the abnormality of fetal chromosomes noninvasively anddirectly becomes an important study topic. In 2007, Professor LO YukMing Dennis and his colleagues proved that the percentage of mutationsite of placenta specific gene 4 in maternal plasma mRNA could be usedfor judging whether the fetus has chromosome 21 which was a triploid(4). The percentage of mutation site is at the same time used forjudging whether chromosome 18 is a triploid (5). Its limitation is inthat the mutation site is not common in the crowd, so these methods areonly suitable for a part of crowd. During the same period, digital PCR(dPCR) is used for detecting the triploid of fetal chromosome (6), (7).The digital PCR has the advantage of independency of any mutation site,but its accuracy is insufficient, and requires many blood samples, whichincreases sampling difficulty.

In recent years, the above problems have been solved by rapidlydeveloped high-throughput DNA sequencing techniques. These techniquesinclude Genome Analyzer of IIlumina (8), SOLiD of Life Technologies (9)and Heliscope of Helicos (10), by which hundreds of millions or evenbillions of sequences can be detected once. When these techniques areused for detecting DANs in maternal plasma, the change of the number ofchromosomes of trace amounts of fetal DNA in plasma can be detected(11), (12), (13). But due to high sequencing cost, these techniques havenot been used commonly. At the same time, there is still an unsolvedproblem of detecting the change of partial copy number of fetalchromosomes from maternal plasma. It is advantageous to detect thechange of copy number of fetal chromosomes from maternal plasma byhigh-throughput sequencing, but this technique is expensive in cost andcannot be popularized. Moreover, the sequencing Coefficient Of Variation(CV) is high, and the detecting accuracy and stability also needs to beimproved. The sequencing CV also decides that this method is onlysuitable for a few chromosomes, such as chromosome 21, chromosome 18,and unsuitable for detecting the change of partial copy number ofchromosomes.

It is very expensive and difficult to detect the change of the number ofchromosomes by high-throughput sequencing, the main reason is that thecontent of fetal DNA in maternal plasma is low, only 5% when it is low,in particular during early fetal development. Most DNA in maternalplasma is maternal DNA. The background of maternal DNA easilyencompasses the change of the number of fetal chromosomes or partialcopy number. Therefore, the method for separating the pregnant women andfetal DNA becomes the subject studied for years with little progress. Asuccessful method should belong to histone separation method invented byBaylor Medical College (14). The quantity of DNA separated is verysmall, so the method is only suitable for detecting the mutation site,unsuitable for detecting the change of the copy number of chromosomes.

SUMMARY OF THE INVENTION

In view of the above problems in the method for detecting the copynumber of fetal chromosomes, the inventor designs a kit, a device and amethod for detecting the whole or partial copy number of fetalchromosomes effectively at low cost.

The invention is based on the following facts: the inventor finds thatthe GC contents of the DNA segments from each chromosome respectivelyhave linear relationships with the ratios of the DNA segments from eachchromosome to the total DNA segments, the above phenomena may be relatedto the detection method, the linear relationship can be represented byy=ax+b, where y represents the GC content of the DNA segment from to thechromosome to be detected, x represents the ratio of the number of theDNA segments from the chromosome to be detected to the total DNA, a andb are constants, a and b may be different values for differentchromosomes, the ratio can be corrected according to the GC content ofthe DNA segments from the chromosomes to be detected, and the variationof the corrected ratio of the DNA segments from the chromosomes to bedetected in the sample to be detected is calculated, and the copy numberof the chromosomes to be detected is determined according to degree ofvariation. By correcting the GC content, many false negative resultsthat cannot be detected only by judgment method of the ratio of the DNAsegments of each chromosome to total DNA segments can be detectedeffectively. The specific experiments are taken as evidences in thedetailed description of the embodiments.

Additionally, as reported in the document (15), most fetal DNA inmaternal plasma are 100 bp to 250 bp segments, particularly in the greatmajority of 150 bp to 170 bp. Although only a tiny part of maternal DNAare distributed in the segment range, the DNA segments of more than 250bp basically belong to maternal DNA. The inventor finds that, althoughthe reason is unknown, the ratio of the DNA segments from eachchromosome to the total DNA segments is uniformly distributed withlength at any point or in any interval within the range of 100 bp to 250bp, i.e., each chromosome is with length at any point within the rangeof 100 bp to 250 bp, such as 110 bp or 167 bp (the quantity of DNA inthe site is the most), the ratio with the total DNA represents the ratioof other points, thus representing the ratio of each chromosome to allDNA within the range of 100 bp to 250 bp. Through sequencing the DNAwith length at any point or in any interval within the range of 100 bpto 250 bp in the DNA, the sequencing results of all DNA with length atany point or in any interval within the range of 100 bp to 250 bp in theDNA are compared with the genomic sequence map to determine whichchromosome each DNA segment of the DNA sequence of all DNA or in anyinterval within the range of 100 bp to 250 bp in the DNA comes from andthe length of each DNA segment; the ratio of the number of the DNAsegments from the chromosomes to be detected in all DNA with length atany point or in any interval within the range of 100 bp to 250 bp in thesame sample to all DNA segments with length at any point or in anyinterval within the range of 100 bp to 250 bp is to calculated to obtainthe ratio of each fetal chromosomes to the total DNA. This greatlyreduces the detecting results. The ratio is corrected according to theGC content of the DNA segments from the chromosomes to be detected inconjunction with the above GC-based correction method, and the variationof the corrected ratio of the DNA segments from the chromosomes to bedetected in the sample to be detected is calculated to determine thecopy number of the chromosomes to be detected according to degree ofvariation.

At the same time, the inventor finds that, similarly during thedevelopment of tumors, the same thing happens in the blood from patientas in the maternal blood during the development of tumors, i.e., in theblood of tumor patient, DNA of free tumor cells can be detected. Thelinear relationship between the GC content of the DNA segments from eachchromosome measured with the method of the invention with the ratios ofthe DNA segments from each chromosome to the total DNA segments issimilarly suitable for detecting aneuploidy of tumor cells. Moreover,DNA of free tumor cells in plasma are present in the form ofnucleosomes, so they are mostly 100 bp to 250 bp segments, the ratio ofthe DNA segments from each chromosome to the total DNA segments isuniformly distributed with length at any point or in any interval withinthe range of 100 bp to 250 bp, i.e., each chromosome is with length atany point within the range of 100 bp to 250 bp, thus representing theratio of each chromosome to all DNA within the range of 100 bp to 250bp. Therefore, the kit, the device and the method of the invention arealso suitable for detecting the copy number of tumor cell chromosomes orpartial chromosomes.

Based on the above findings, the inventor provides a kit, a device and amethod for detecting the copy number of fetal chromosomes or tumor cellchromosomes or partial chromosomes non-invasively and economically.

A kit for detecting the copy number of fetal chromosomes or tumor cellchromosomes provided by the invention includes: an instrument forcollecting blood from a pregnant women or a tumor patient; an instrumentfor separating blood cells from plasma in blood; a reagent and aninstrument for extracting Deoxyribonucleic Acids (DNA) in the plasma; areagent and an instrument for separating the DNA with a physical methodaccording to the size of the DNA segments; and a reagent and aninstrument for sequencing DNA with length with length at any point or inany interval within the range of 100 bp to 250 bp.

Preferably, the fetal chromosomes or tumor cell chromosomes are thewhole chromosomes or partial chromosomes.

Preferably, the kit of the invention further includes: a reagent and aninstrument for preparing all DNA into a sequencing library.

Preferably, the kit of the invention further includes: a reagent and aninstrument for performing PCR amplification of the DNA extracted fromplasma or the sequencing library.

Preferably, the DNA with length at any point or in any interval withinthe range of 100 bp to 250 bp is the 150 bp-170 bp DNA, more preferably,is the 167 bp DNA.

Another kit for detecting the copy number of fetal chromosomes or tumorcell chromosomes provided by the invention includes: an instrument forcollecting blood from a pregnant women or a tumor patient; an instrumentfor separating blood cells from plasma in blood; a reagent and aninstrument for extracting DNA from the plasma; a reagent and aninstrument for preparing the DNA into a sequencing library; and areagent and an instrument for sequencing the DNA, wherein the fetalchromosomes or tumor cell chromosomes are the whole chromosomes orpartial chromosomes.

Preferably, the kit of the invention further includes: a reagent and aninstrument for performing PCR amplification on the DNA extracted fromplasma.

A device for detecting the copy number of fetal chromosomes or tumorcell chromosomes provided by the invention includes: a detecting module,which is used for sequencing DNA in a sample of maternal plasma orplasma of tumor patient, wherein the sequencing includes preparing allDNA in the sample of maternal plasma or plasma of tumor patient into asequencing library; a comparison module, which is used for comparing asequencing result of the DNA with a genomic sequence map to determinewhich chromosome each DNA sequence comes from and the length of each DNAsequence; a calculating module, which is used for calculating the ratioof the number of DNA segments from the chromosomes to be detected to thetotal number of DNA segments in the same sample, correcting the ratioaccording to a GC content of the DNA segments from the chromosomes to bedetected, and calculating the variation of the corrected ratio of theDNA segments from the chromosomes to be detected in a sample to bedetected, and determining the copy number of the chromosomes to bedetected according to degree of variation; and an output module, whichis used for outputting the copy number of the chromosomes to bedetected.

Preferably, the calculating module in the device of the inventioncorrects the ratios of the chromosomes 2, 3, 4, 5, 6, 7, 8, 12, 13, 18and X according to the following function: the GC contents of the DNAsegments from the chromosomes 2, 3, 4, 5, 6, 7, 8, 12, 13, 18 and Xrespectively have linear relationships with the ratios of the DNAsegments from each chromosome to the total DNA segments, the linearrelationship can be represented by y=ax+b, where y represents the GCcontent of the DNA segment from the chromosome to be detected, xrepresents the ratio of the number of the DNA segments from thechromosome to be detected to the total DNA, a and b are constants, and ais negative.

Preferably, the linear relationship between the GC contents of the DNAsegments from the chromosomes 2, 3, 4, 5, 6, 7, 8, 12, 13, 18 and X andthe ratios of the DNA segments from each chromosome to the total DNAsegments is as shown in Table 1.

Preferably, the calculating module in the device of the inventioncorrects the ratios of the chromosomes 1, 9, 10, 11, 15, 16, 17, 19, 20,21 and 22 according to the following function: the GC contents of theDNA segments from the chromosomes 1, 9, 10, 11, 15, 16, 17, 19, 20, 21and 22 respectively have linear relationships with the ratios of the DNAsegments from each chromosome to the total DNA segments, the linearrelationship can be represented by y=ax+b, where y represents the GCcontent of the DNA segment from the chromosome to be detected, xrepresents the ratio of the number of the DNA segments from thechromosome to be detected to the total DNA, a and b are constants, and ais positive.

Preferably, the linear relationship between the GC contents of the DNAsegments from the chromosomes 1, 9, 10, 11, 15, 16, 17, 19, 20, 21 and22 and the ratios of the DNA segments from each chromosome to the totalDNA segments is as shown in Table 2.

Preferably, the sequencing includes the process of preparing all DNA inthe sample of maternal plasma or plasma of tumor patient into asequencing library.

Preferably, sequencing the DNA in the sample of maternal plasma orplasma of tumor patient is performed by paired-end short sequencesequencing, single-end long sequence sequencing or single-end shortsequence sequencing.

Preferably, the fetal chromosomes or tumor cell chromosomes are thewhole chromosomes or partial chromosomes.

Preferably, the PCR amplification of the DNA in the sample of maternalplasma or plasma of tumor patient is performed DNA before sequencing.

Preferably, in the device of the invention, the calculating module isused for calculating the ratio of the number of the DNA segments fromthe chromosomes to be detected in all DNA with length at any point or inany interval within the range of 100 bp to 250 bp in the same sample tothe total number of all DNA segments with length at any point or in anyinterval within the range of 100 bp to 250 bp, correcting the ratioaccording to the GC content of the DNA segments from the chromosomes tobe detected in all DNA with length at any point or in any intervalwithin the range of 100 bp to 250 bp, and calculating the variation ofthe corrected ratio of the DNA segments from the chromosomes to bedetected in the sample to be detected, and determining the copy numberof the chromosomes to be detected according to degree of variation.

Preferably, all DNA with length at any point or in any interval withinthe range of 100 bp to 250 bp in the sample of maternal plasma or plasmaof tumor patient are prepared into the sequencing library.

Preferably, the PCR amplification of the DNA in the sample of maternalplasma or plasma of tumor patient is performed DNA before or after beingprepared into the sequencing library.

Preferably, the DNA with length at any point or in any interval withinthe range of 100 bp to 250 bp is the 150 bp-170 bp DNA, more preferablythe 167 bp DNA.

A method for detecting the copy number of fetal chromosomes or tumorcell chromosomes provided by the invention includes the following steps:collecting maternal plasma or plasma of tumor patient; separating theplasma from blood cells in blood; preparing DNA in the plasma into asequencing library; sequencing the DNA sequencing library; comparing asequencing result with a genomic sequence map to determine whichchromosome each DNA sequence comes from and the length of each DNAsegment; and calculating the ratio of the DNA segments from thechromosomes to be detected to the total number of the DNA segments bysequencing and comparison results of DNA, correcting the ratio accordingto a GC content of the DNA segments from the chromosomes to be detected,and calculating the variation of the corrected ratio of the DNA segmentsfrom the chromosomes to be detected in a sample to be detected, anddetermining the copy number of the chromosomes to be detected accordingto degree of variation.

Preferably, the method of the invention further includes correcting theratios of the chromosomes 2, 3, 4, 5, 6, 7, 8, 12, 13, 18 and Xaccording to the following function: the GC contents of the DNA segmentsfrom the chromosomes 2, 3, 4, 5, 6, 7, 8, 12, 13, 18 and X respectivelyhave linear relationships with the ratios of the DNA segments from eachchromosome to the total DNA segments, the linear relationship can berepresented by y=ax+b, where y represents the GC content of the DNAsegment from the chromosome to be detected, x represents the ratio ofthe number of the DNA segments from the chromosome to be detected to thetotal DNA, a and b are constants, and a is negative.

Preferably, the linear relationship between the GC contents of the DNAsegments from the chromosomes 2, 3, 4, 5, 6, 7, 8, 12, 13, 18 and X andthe ratios of the DNA segments from each chromosome to the total DNAsegments is as shown in Table 1.

Preferably, the method of invention further includes correcting theratios of the chromosomes 1, 9, 10, 11, 15, 16, 17, 19, 20, 21 and 22according to the following function: the GC contents of the DNA segmentsfrom the chromosomes 1, 9, 10, 11, 15, 16, 17, 19, 20, 21 and 22respectively have linear relationships with the ratios of the DNAsegments from each chromosome to the total DNA segments, the linearrelationship can be represented by y=ax+b, where y represents the GCcontent of the DNA segment from the chromosome to be detected, xrepresents the ratio of the number of the DNA segments from thechromosome to be detected to the total DNA, a and b are constants, and ais positive.

Preferably, the linear relationship between the GC contents of the DNAsegments from the chromosomes 1, 9, 10, 11, 15, 16, 17, 19, 20, 21 and22 and the ratios of the DNA segments from each chromosome to the totalDNA segments is as shown in Table 2.

Preferably, the method includes calculating the ratio of the number ofthe DNA segments from the chromosomes to be detected in all DNA withlength at any point or in any interval within the range of 100 bp to 250bp in the same sample to the total number of all DNA segments withlength at any point or in any interval within the range of 100 bp to 250bp only by sequencing and comparison results of all DNA with length atany point or in any interval within the range of 100 bp to 250 bp,correcting the ratio according to the GC content of the DNA segmentsfrom the chromosomes to be detected in all DNA with length at any pointor in any interval within the range of 100 bp to 250 bp, and calculatingthe variation of the corrected ratio of the DNA segments from thechromosomes to be detected in the sample to be detected, and determiningthe copy number of the chromosomes to be detected according to degree ofvariation.

Preferably, sequencing the DNA sequencing library is performed bypaired-end short sequence sequencing, single-end long sequencesequencing or single-end short sequence sequencing.

Preferably, the DNA with length at any point or in any interval withinthe range of 100 bp to 250 bp is the 150 bp-170 bp DNA, more preferablythe 167 bp DNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an image obtained by 1% agarose gel electrophoresis afterpreparing the DNA in maternal plasma into a library for paired-endsequencing. Left channel is an image of DNA 100 bp marker, right channelis an image of library for paired-end sequencing, and the most obviousstripe is located at about 280 bp, containing 120 bp connecting primers.

FIG. 2 is a distribution graph of DNA segment size of sample G356.

FIG. 3 is a distribution graph of DNA segment from X chromosome in acompared sample G356 made according to the segment size.

FIG. 4 is a distribution graph of calculated and actually measured DNAsegments of X chromosome of G356 according to the segment size, in whichthe line with asterisk represents a graph obtained by multiplying thetotal number of DNA sequences of sample G356 by percent of X chromosome,circle represents the sequence number of X chromosomes actuallymeasured. As shown in figure, the DNA segment distribution of G356 Xchromosome calculated is the same as the actually measured distributionessentially, these two lines are basically consistent with each other.

FIGS. 5A-5H are standard curves drawn according to the GC contents ofthe DNA segments from the chromosomes to be detected in G356, G397(repeated for 8 times), G426, G735, G756, G760, G763, G770, G778, G779,G780, G781, G824 and G825 and the ratios of the DNA segments from thechromosomes to be detected to the total DNA segments, in which samplesG356, G397, G426, G735, G756, G760, G763, G770, G778, G779, G780, G781,G824 and G825 are identified as normal female fetus samples [46, XX]through amniocentesis by chromosome karyotype in Xiangya Medical Collegeof Central South University. X axis presents the ratio of the number ofthe DNA segments from the chromosomes to be detected to the total DNAsegments, Y axis represents the GC content of DNA segments from thechromosomes to be detected. Each of 5A-5F shows standard curves of threechromosomes, orderly showing standard curves of the 1-th to the 18-thchromosomes, FIG. 5G shows a standard curve of the 19-th to the 22-thchromosomes, FIG. 5H shows a standard curve of X chromosome. Thefunctions of standard curves of each chromosome are as shown in Table 1and Table 2.

FIG. 6 is a schematic diagram of a sample of trisomy 13 detected by themethod of the invention, in which X axis represents the ratio of thenumber of DNA segments from chromosome 13 to the total DNA segments, Yaxis represents the GC content of the DNA segments from chromosome 13.The diamond on the X axis shows the ratio of the number of the DNAsegments from chromosome 13 to the total DNA segments in each sample,the rectangle indicated by arrow represents a sample of trisomy 13detected after correcting the GC content of DNA segments from chromosome13. If the correction of the GC content of DNA segments from thechromosomes to be detected of the present invention does not beperformed, three abnormal samples indicated by arrow cannot be foundonly by the ratio of the DNA segments from chromosome 13 to the totalDNA segments, and there would be a false negative result.

FIG. 7 is a schematic diagram of a sample of trisomy 18 detected by themethod of the invention, in which X axis represents the ratio of thenumber of the DNA segments of chromosome 18 to the total DNA segments, Yaxis represents the GC content of the DNA segments from chromosome 18.The diamond on the X axis shows the ratio of the number of the DNAsegments from chromosome 18 to the total DNA segments in each sample,the rectangle indicated by arrow represents a sample of trisomy 18detected after correcting the GC content of DNA segments from chromosome18. If the correction of the GC content of DNA segments from thechromosomes to be detected of the present invention does not beperformed, it is can be only found that 5 samples in which the ratio ofthe DNA segment from chromosome 18 to the total DNA segments is largerthan or equal to 0.031 is the trisomy 18, and it cannot be found thattwo samples with a ratio of about 0.03 are abnormal, only by the ratioof the DNA segments from chromosome 18 to the total DNA segments, withrespect to the two samples, there would be a false negative result.

FIG. 8 is a schematic diagram of a sample of X monosomy or trisomydetected by the method of the invention, in which X axis represents theratio of the number of the DNA segments of X chromosome to the total DNAsegments, Y axis represents the GC content of the DNA segments from Xchromosome. The diamond on the X axis shows the ratio of the number ofthe DNA segments from X chromosome to the total DNA segments in eachsample, the rectangle indicated by arrow on the left of standard curverepresents a sample of X monosomy detected after correcting the GCcontent of DNA segments from X chromosome, the rectangle indicated byarrow on the right of standard curve represents a sample of X trisomydetected after correcting the GC content of DNA segments from Xchromosome. If the correction of the GC content of DNA segments from thechromosomes to be detected of the present invention does not beperformed, one sample of X trisomy can only be found and the sample of Xmonosomy cannot be found easily only by the ratio of the DNA segmentsfrom X chromosome to the total DNA segments, and there would be a falsenegative result.

FIGS. 9A-9B are schematic diagrams of a sample of trisomy 21 detected bythe method of the invention, in which X axis represents the ratio of thenumber of the DNA segments of chromosome 21 to the total DNA segments, Yaxis represents the GC content of the DNA segments from chromosome 21.The diamond on the X axis shows the ratio of the number of the DNAsegments from chromosome 21 to the total DNA segments in each sample,the rectangle indicated by arrow represents a sample of trisomy 21detected after correcting the GC content of DNA segments from chromosome18. If it is can be only found that 4 samples in which the ratio of theDNA segment from chromosome 21 in FIG. 9 to the total DNA segments isthe maximum is the trisomy 21, and it cannot be found that the sampleswith a ratio of about 0.0138 are abnormal, only by the ratio of the DNAsegments from chromosome 18 to the total DNA segments rather than bycorrecting the GC content of DNA segments from the chromosomes to bedetected, with respect to the samples, there is a false negative result.

DETAILED DESCRIPTION OF THE EMBODIMENTS

It should be noted that, without contradiction, the embodiments in theapplication and the characteristics in the embodiments can be combined.The invention is described below with reference to drawings inconjunction with embodiments. The drawings of the invention and theembodiments are only used for explaining the invention, and not intendedto limit the invention.

Term Definition

Paired-end short sequence refers to a sequence of less than 50 bp nextto 5′-terminal connecting primer and a sequence of less than 50 bp nextto 3′-terminal connecting primer. Preferably, paired-end short sequencerefers to a sequence of not more than 36 bp next to 5′-terminalconnecting primer and a sequence of not more than 36 bp next to3′-terminal connecting primer.

Single-end short sequence refers to a sequence of less than 50 bp nextto 5′-terminal connecting primer or a sequence of less than 50 bp nextto 3′-terminal connecting primer. Preferably, single-end short sequencerefers to a sequence of not more than 36 bp next to 5′-terminalconnecting primer or a sequence of not more than 36 bp next to3′-terminal connecting primer.

Single-end long sequence refers to a sequence of more than 99 bp next to5′-terminal connecting primer or a sequence of more than 99 bp next to3′-terminal connecting primer.

Paired-end sequencing refers to testing the sequence at both ends of thesequence.

Single-end sequencing refers to testing the sequence at one end of thesequence.

DNA cluster refers to multiple DNA molecules formed by amplifying oneDNA molecule and located in one fixed surface area. In the embodimentsof the application, DNA cluster refers to about 1000 DNA moleculesformed by amplifying one DNA molecule and located within 1 squaremicron.

Emulsion Polymerase Chain Reaction (PCR) refers to performing PCRreaction by placing PCR reactants including PCR template DNA, PCRprimer, PCR polymerase and free bases within one oil droplet. Usually,within one oil droplet, the template of emulsion PCR has only one DNAmolecule.

GC content refers to the ratio of the number of guanine and cytosine tothe total number of all bases in nucleic acids or deoxyribonucleicacids.

Embodiment 1 A Method for Detecting the Copy Number of Fetal Chromosomes

Step 1: Collecting maternal blood to prepare plasma.

In the embodiment, 14 maternal blood samples are extracted, samplecodes: G356, G397, G426, G735, G756, G760, G763, G770, G778, G779, G780,G781, G824 and G825, all identified as normal female fetus samples [46,XX] by Professor Wu Lingqian of Xiangya Medical College of Central SouthUniversity through amniocentesis by chromosome karyotype. The detecteddata of the above samples are used for drawing a standard curve, and theabove samples are also used as standard samples. Plasma samples fromwhich blood cells are removed are obtained after centrifuging the bloodsamples at high speed, and each sample has a plasma volume of about 1ml.

Step 2: Extracting plasma DNA.

DNA in the plasma is extracted by using DNA extraction kit produced byQiagen (product number: 57704).

Step 3: Preparing the plasma DNA into a sequencing library.

The plasma DNA can be prepared into a library for paired-end shortsequence sequencing, single-end long sequence sequencing or single-endshort sequence sequencing. The process for preparing the library forpaired-end short sequence sequencing is as follows.

The extracted DNA is end-blunted and subjected to 5′-terminalphosphorylation: 30 μl of DNA, 45 μl of pure water, 10 μl of T4 DNAligase buffer with 10 mM ATP, 4 μl of 10 mM dNTP Mix, 5 μl of T4 DNApolymerase, 1 μl of Klenow enzyme and 5 μl of T4 PNK are treated in awarm bath for 30 min at 20 after mixing (the reagents are provided byIllumina sample preparation kit PE-102-1001). The DNA is purified byQIAGEN QIAquick PCR purification kit (part #28104) after warm bathtreatment.

Suspending A at terminal: the resulting product from the above step isdissolved in 32 μl of buffer, 5 μl of Klenow buffer, 10 μl of 1 mM dATPand 3 μl of Klenow Exo are added into the mixture, and kept for 30 minat 37 (the reagents are provided by Illumina sample preparation kitPE-102-1001), the resulting product is purified by QIAGEN MinElute PCRpurification kit (part #28004).

Connecting: the DNA is dissolved in 10 μl of buffer, 2×25 μl of DNAligase buffer, 10 μl of PE Adapter Oligo Mix and 5 μl of DNA ligase areadded to the mixture, and kept for 15 min at 20 (the reagents areprovided by Illumina sample preparation kit PE-102-1001). The DNA ispurified by QIAGEN QIAquick PCR purification kit (part #28104) afterwarm bath treatment.

FIG. 1 is a gel electrophoresis image, in which the DNA in a sample areprepared into a library for paired-end sequencing, the DNA in thelibrary are subjected to electrophoresis by 1% of agarose gel, the mostobvious stripe is located at 280 bp, due to containing 120 bp linkerprimers, the main DNA segments in the maternal plasma mainly focus atabout 160 bp.

Preferably, PCR amplification can also be performed on the library forpaired-end sequencing: 1 μl of DNA, 22 μl of pure water, 1 μl of PE PCRprimer PE 2.0, 1 μl of PE to PCR primer PE 1.0 and 2× Phusion DNApolymerase (Finnzymes Oy) (the reagents are provided by Illumina samplepreparation kit PE-102-1001). The DNA is amplified through PCRinstrument, the procedure is 98, 30 s, 98, 40 s, 65, 30 s, 72, 30 s, 12cycles in total, 72, 5 min.

Step 4: Sequencing the DNA sequencing library.

According to different libraries prepared in Step 3, paired-end shortsequence sequencing, single-end long sequence sequencing or single-endshort sequence sequencing can be performed respectively. The process forperforming paired-end short sequence sequencing is as follows.

Single DNA molecule in DNA paired-end sequencing library is preparedinto DNA cluster by cBot instrument of Illumina, this step also can be astep of changing single DNA molecule into polymolecule in a droplet byemulsion PCR. The generated DNA cluster or DNA droplet obtained byemulsion PCR is subjected to paired-end sequencing in Genome Analyzer orHiSeq2000 sequencer of Illumina. The process is automatically finishedby the instrument itself.

Alternatively, the generated DNA cluster or DNA droplet obtained byemulsion PCR is subjected to single-end long sequence sequencing inGenome Analyzer or HiSeq2000 of Illumina, or SOLiD sequencer of LifeTechnologies. The sequencing steps and reaction conditions in GenomeAnalyzer or HiSeq2000 of Illumina, or SOLiD sequencer of LifeTechnologies are the same as above described.

Step 5: Determining which chromosome the DNA segment in the plasma comesfrom and determining whether the copy number of the chromosomes to bedetected is normal.

After performing paired-end short sequence sequencing of the DNA library(alternatively, measuring single-end long sequence sequencing orsingle-end short sequence sequencing), when each 36 bp base sequence ateach end of one DNA segment is known, the sequences on both ends can becompared with human genome standard sequence 37.1(http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/human/data/?build=37),which is also called hg19 to determine the respective position of thesequences at both ends on the chromosome. The distance between thesequences at both ends is the length of the DNA segment, at the sametime, the chromosomal position of the sequences at both ends determinewhich chromosome the DNA segment comes from.

FIG. 2 is a distribution graph of DNA segment size of sample G356determined according to the above method, from which short DNA in thematernal plasma mainly focus on between 100 bp and 220 bp.

FIG. 3 is a distribution graph of DNA segment from X chromosome in acompared sample G356 made according to the segment size. The obtainedgraph is substantially the same as FIG. 2.

Step 6: Calculating the ratio of the number of DNA segments from thechromosomes to be detected to all DNA segments in the DNA in the samesample, correcting the ratio according to the GC content of the DNAsegments from the chromosomes to be detected, and calculating thevariation of the corrected ratio of the DNA segments from thechromosomes to be detected in a sample to be detected, and determiningthe copy number of the chromosomes to be detected according to degree ofvariation.

Preferably, the method includes calculating the ratio of the number ofthe DNA segments from the chromosomes to be detected in all DNA withlength at any point or in any interval within the range of 100 bp to 250bp in the same sample to the total number of all DNA segments withlength at any point or in any interval within the range of 100 bp to 250bp by sequencing and comparison results of all DNA with length at anypoint or in any interval within the range of 100 bp to 250 bp DNAsequence length, correcting the ratio according to the GC content of theDNA segments from the chromosomes to be detected, and calculating thevariation of the corrected ratio of the DNA segments from thechromosomes to be detected in the sample to be detected, and determiningthe copy number of the chromosomes to be detected according to degree ofvariation.

In experiments, it is found that the accuracy of experimental result canbe improved by sequencing and comparison results of all DNA with lengthat any point or in any interval within the range of 100 bp to 250 bp DNAsequence length.

The specific algorithm is as follows: Using standard samples,calculating the ratio of the DNA segments from the chromosomes to bedetected in the same sample to all DNA segments, obtaining the GCcontent of the DNA segment from the chromosomes to be detected accordingto the sequencing results; drawing a standard curve of GC content (Yaxis) and the percentage (X axis) of each chromosome or partialchromosome to all chromosomes according to the above ratio and GCcontent; correcting the measured ratio of chromosome of sample to bedetected into a fixed GC value (usually arithmetic mean of GC value)according to its own function of the chromosome; and calculating thevariation value Z of the corrected ratio of the DNA segments from thechromosomes to be detected in the sample to be detected, and determiningthe copy number of the chromosomes to be detected according to Z value.

FIGS. 5A-5H are standard curves drawn according to the GC contents ofthe DNA segments from the chromosomes to be detected in measured samplesand the ratios of the DNA segments from the chromosomes to be detectedto the total DNA segments. It can be seen from the figures that the GCcontents of the DNA segments from chromosomes 2, 3, 4, 5, 6, 7, 8, 12,13, 14, 18 and X are respectively in inverse proportion to the ratios ofthe DNA segments from each chromosome to the total DNA segments, thelinear relationship linear relationships with the ratios of the DNAsegments from each chromosome to the total DNA segments, the linearrelationship can be represented by y=ax+b, where y represents the GCcontent of the DNA segment from the chromosome to be detected, xrepresents the ratio of the number of the DNA segments from thechromosome to be detected to the total DNA, a and b are constants, and ais negative. It should be noted that, with respect to differentreference samples, small changes may occur in specific parameters of theformula, but the overall trend is unchanged. The functions ofchromosomes 2, 3, 4, 5, 6, 7, 8, 12, 13, 14, 18 and X are shown in Table1:

TABLE 1 Chromosome No. Function 2 Y = −1274.5X + 154.48 3 Y = −682.68X +88.11 4 Y = −391.42X + 62.075 5 Y = −772.25X + 88.517 6 Y = −729.61X +84.354 7 Y = −2874.7X + 197.28 8 Y = −1599.4X + 122.84 12 Y = −1936.7X +129.32 13 Y = −827.29X + 67.049 14 Y = −933.9X + 74.22 18 Y = −1946.4X +97.323 X Y = −749.77X + 71.33

The GC contents of the DNA segments from chromosomes 1, 9, 10, 11, 15,16, 17, 19, 20, 21 and 22 are respectively in proportion to the ratiosof the DNA segments from each chromosome to the total DNA segments, thelinear relationship can be represented by y=ax+b, where y represents theGC content of the DNA segment from the chromosome to be detected, xrepresents the ratio of the number of the DNA segments from thechromosome to be detected to the total DNA, a and b are constants, and ais positive.

TABLE 2 Chromosome No. Function 1 Y = 909.17X − 29.372 9 Y = 2211.3X −45.632 10 Y = 1886.8X − 51.026 11 Y = 1255X − 15.714 15 Y = 1775.8X −10.124 16 Y = 797.17X + 23.883 17 Y = 560.83X + 31.227 19 Y = 513.79X +43.088 20 Y = 1006.7X + 22.818 21 Y = 7298X − 51.083 22 Y = 596.24X +43.346

Then, the measured ratio of chromosome of sample to be detected iscorrected into a fixed GC value (usually arithmetic mean of GC value)according to its own function of the chromosome.

For example, it can be seen from the function y=ax+b represented by GCcontents of the DNA segments from each chromosome and the ratios of theDNA segments from each chromosome to the total DNA segments that, theratio x of the DNA segments from each chromosome to the total DNAsegments in each sample can be corrected through the function accordingto GC value. The correction formula can be

${z = {\overset{\_}{e} - \frac{\left( {y - \hat{y}} \right)}{a}}},$

where x is the corrected ratio of the DNA segments from each chromosometo the total DNA segments; ē is the ratio of the specified chromosomesegment to the total DNA segments in the detected sample; y is the GCcontent of the specified chromosome segment in the sample to bedetected; ŷ is arithmetic mean GC value of the chromosome segment indetected standard sample (known normal sample, such as 14 known normalstandard samples detected in Embodiment 1). a is the slope of thiscurve. For the function of each chromosome, see Table 1 and Table 2; Fordifferent samples, e and ŷ are measured values of this test, which varyalong with different samples to be detected.

Through calculating average values of these corrected X values andstandard errors, value Z is obtained: Z=(corrected ratio×of sample to bedetected−corrected average value of ratio of each standardsample)/standard error. The variation of the corrected ratio x in eachsample to be detected is calculated, and the copy number of thechromosomes to be detected or partial chromosomes to be detected isdetermined according to value Z of variation. In general, the absolutevalue of Z being smaller than 3 is considered as normal detection error,and the absolute value of Z being bigger than 3 is considered to beabnormal.

The following Tables 3, 4 and 5 show detection and correction results ofchromosomes 13, 18 and 21 calculated by detecting samples G356, G397(repeated for 8 times), G426, G735, G756, G760, G763, G770, G778, G779,G780, G781, G824 and G825 with the method of Embodiment 1.

TABLE 3 Correction table of chromosome 13 Sample X Y Corrected X G3560.032128056 40.40838593 0.0312599 G397L1 0.031796287 40.741293670.031330538 G397L2 0.031829498 40.74485255 0.031368051 G397L30.031300856 41.02955526 0.031183548 G397L4 0.031461675 40.975273140.031278753 G397L5 0.030861045 41.52415988 0.031341599 G397L60.031076999 41.18977275 0.031153357 G397L7 0.030740791 41.54455850.031246002 G397L8 0.031068253 41.29163215 0.031267736 G426 0.03211672240.65542488 0.031547179 G735 0.030833804 41.76349467 0.031603657 G7560.032145341 40.54153095 0.031438126 G760 0.032505467 40.227542760.031418714 G763 0.03135526 41.028428 0.03123659 G770 0.02932472542.84226241 0.031398557 G778 0.030580783 41.74167735 0.031324265 G7790.030604185 41.75611131 0.031365114 G780 0.03115125 41.497627970.031599733 G781 0.030039316 42.09062396 0.031204592 G824 0.03273105539.92248585 0.031275559 G825 0.032354852 40.14196058 0.03116465 Average0.03133363 41.1266026 0.03133363 value Standard 0.000853052 0.7140977040.000131819 error Slope −827.2898903

TABLE 4 Correction table of chromosome 18 Sample X Y Corrected X G3560.028355 42.32462 0.028009403 G397L1 0.028042 42.66689 0.027871871G397L2 0.028211 42.5936 0.028003058 G397L3 0.027866 42.92468 0.027828101G397L4 0.028163 42.8853 0.028105316 G397L5 0.027723 43.42382 0.027941782G397L6 0.027887 43.01112 0.027893673 G397L7 0.027615 43.522030.027884151 G397L8 0.027718 43.18862 0.027815666 G426 0.028085 42.344710.027749591 G735 0.027524 43.74306 0.027906323 G756 0.028028 42.565830.027805722 G760 0.028321 42.13679 0.02787838 G763 0.027859 42.817590.027766269 G770 0.027028 44.70943 0.027907267 G778 0.02775 43.556820.028036728 G779 0.02766 43.52168 0.027929435 G780 0.027819 43.347230.027998124 G781 0.027553 43.88948 0.028010923 G824 0.02851 41.75960.027874031 G825 0.028393 42.02757 0.027894578 Average 0.02791 42.998120.027910019 value Standard 0.000353 0.710198 0.0000921681 error Slope−1946.44

TABLE 5 Correction table of chromosome 21 Sample X Y Corrected X G3560.01313 44.40818 0.013271072 G397L1 0.013154 44.93124 0.013223969 G397L20.013215 44.89741 0.013289069 G397L3 0.013191 45.45013 0.013189714G397L4 0.013192 45.39349 0.013197949 G397L5 0.013315 46.104430.013224459 G397L6 0.013301 45.56648 0.013283986 G397L7 0.01334746.32729 0.013225833 G397L8 0.01339 45.78404 0.013342788 G426 0.01315744.51204 0.013283828 G735 0.013269 46.54507 0.013117886 G756 0.01321244.89562 0.013286433 G760 0.013046 44.22742 0.01321199 G763 0.01318145.2873 0.013202366 G770 0.01351 47.67432 0.013203677 G778 0.01323545.93867 0.0131666 G779 0.013146 46.1604 0.013047278 G780 0.01317745.80678 0.013126693 G781 0.013333 46.55558 0.013180262 G824 0.01308143.62277 0.0133298 G825 0.013164 44.15792 0.013339786 Average 0.01322645.44031 0.013225973 value Standard 0.000109 0.973152 0.0000762249 errorSlope 7298.004

Note that, the above detection and calculation method is not onlysuitable for detecting the copy number abnormality of the wholechromosome, but also suitable for detecting the copy number abnormalityof the partial chromosome.

Below is an Embodiment in which the Copy Number of Fetal Chromosome 13in a Sample to be Detected is Detected.

As described above, a total of 15 maternal blood samples are extracted,plasma samples from which blood cells are removed are obtained aftercentrifuging the blood samples at high speed, and each sample has aplasma volume of about 1 ml. The sample code is G352, G362, G372, G383,G397 (repeated for 8 times), G402, G409, G415, G424, G445, G440, G503,G588, G735 and G783. The above samples are collected by Professor WuLingqian from Xiangya Medical College of Central South University.

The trisomy 13 which is possibly present is detected. The detectionresult is verified by standard curve of chromosome 13 obtained above. Asshown in FIG. 6, in the absence of GC correction (marked with diamond onthe X axis in the figure), it is impossible to distinguish trisomy 13samples G445, G352 and G402 (samples marked with 3 circles on the xaxis) from normal samples (samples without marks on the x axis).However, in the presence of GC correction (represented by rectangle inthe figure), it is possible to clearly distinguish trisomy 13 samplesG445, G352 and G402 (samples marked with 3 arrows in the figure) fromnormal samples (other samples represented by rectangle in the figure).The GC correction result is the same as the result identified byProfessor Wu Lingqian from Xiangya Medical College of Central SouthUniversity through amniocentesis by chromosome karyotype. As a result,the correction of GC content can be used for detecting trisomy 13 toreduce the occurrence of false negative result.

The following Table 6 only illustrates the detection and calculationresult of part of samples to be detected, and does not illustrate theresult of other samples.

TABLE 6 Calculation table of Z value of corrected chromosome 13Karyotype Corrected Sample result Corrected X Z value G445 47, XX, +130.033854 19.12307 G352 47, XY, +13 0.033835 18.97637 G402 47, XY, +130.034761 25.99744 G383 46, XY 0.031641 2.331371 G415 47, XX, +180.031497 1.23855 G503 46, XY 0.031504 1.29343 G424 47, XY, +18 0.0315671.771192 Chromosome 13 Corrected X Average value 0.031334 Standard error0.000132

It can be seen from the above table that, the Z value of samples G445,G352, G402 is more than 3, which can be judged as trisomy 13. The Zvalue of other samples is between −3 and +3.

Below is an Embodiment in which the Copy Number of Fetal Chromosome 18in a Sample to be Detected is Detected.

As described above, a total of 16 maternal blood samples are extracted,plasma samples from which blood cells are removed are obtained aftercentrifuging the blood samples at high speed, and each sample has aplasma volume of about 1 ml. The sample code is G362, G372, G383, G397(repeated for 8 times), G407, G409, G415, G424, G442, G432, G445, G440,G595, G588, G735 and G783. The above samples are collected by ProfessorWu Lingqian of Xiangya Medical College of Central South University.

The trisomy 18 which is possibly present is detected. As shown in FIG.7, the detection result is verified by standard curve of chromosome 18obtained above. In the absence of GC correction (marked with diamond onthe X axis in the figure), it is impossible to distinguish some trisomy18 samples (samples marked with 1 back circle on the x axis) from normalsamples (samples without marks on the x axis represented by diamond),which results in false negative samples. Other trisomy 18 samples(samples marked with down arrow on the X axis) can be detected withoutGC correction. However, in the presence of GC correction (represented byrectangle in the figure), it is possible to clearly distinguish alltrisomy 18 samples (samples marked with horizontal arrows in the figure)from normal samples (other samples represented by rectangle in thefigure). The GC correction result is the same as the result identifiedby Professor Wu Lingqian of Xiangya Medical College of Central SouthUniversity through amniocentesis by chromosome karyotype. As a result,the correction of GC content can be used for detecting trisomy 18 toreduce the occurrence of false negative result.

The following Table 7 only illustrates the detection and calculationresult of part of samples to be detected, and does not illustrate theresult of other samples.

TABLE 7 Calculation table of Z value of corrected chromosome 18Karyotype Corrected Sample result Corrected X Z value G424 47, XY, +180.031846 42.7029466 G442 47, XY, +18 0.03259 50.7813669 G432 47, XY, +180.031475 38.6805289 G415 47, XX, +18 0.029786 20.35574454 G595 47, XY,+18 0.031124 34.87420168 G407 47, XY, +18 0.030189 24.72396854 G372 46,XY 0.027704 −2.234074343 G383 46, XY 0.027657 −2.740277188 G362 46, XY0.02795 0.428428385 Chromosome 18 Corrected X Average value 0.027910019Standard error 0.0000921681

It can be seen from the above table that, the Z value of samples G424,G442, G432, G415, G595, G407 is more than 3, which can be judged astrisomy 18. The Z value of other samples is between −3 and +3.

Below is an Embodiment in which the Copy Number of Fetal Chromosome 21in a Sample to be Detected is Detected.

As described above, a total of 14 maternal blood samples are extracted,plasma samples from which blood cells are removed are obtained aftercentrifuging the blood samples at high speed, and each sample has aplasma volume of about 1 ml. The sample code is G267, G387, G393, G376,G397 (repeated for 8 times), G405, G408, G409, G440, G491, G588, G641,G735 and G783. The above samples are collected by Professor Wu Lingqianof Xiangya Medical College of Central South University.

The trisomy 21 (i.e., Down's syndrome sample) which is possibly presentis detected. As shown in FIG. 9A, the detection result is verified bystandard curve of chromosome 21 obtained above. In the absence of GCcorrection (marked with diamond on the X axis in the figure), thetrisomy 21 sample (1 sample marked with black circles and 3 verticalarrows on the x axis) can be detected only by the percentage of thetrisomy 21 in all chromosomes. The difference between one sample (asample marked with black circle on the x axis) and normal sample (othersamples marketed with diamond) is not obvious, which may result in falsenegative samples. However, in the presence of GC correction (representedby rectangle in the figure), it is possible to clearly distinguish alltrisomy 21 samples (samples marked with 4 horizontal arrows in thefigure) from normal samples (other samples represented by rectangle inthe figure). The GC correction result is the same as the resultidentified by Professor Wu Lingqian of to Xiangya Medical College ofCentral South University through amniocentesis by chromosome karyotype.The improvement of the accuracy of trisomy 21 by GC content correctioncan be measured by minimum distance between trisomy 21 and normalsample. As shown in FIG. 9B, the corrected minimum distance d1 is morethan the minimum distance d2 which has not been corrected. As a result,the correction of GC content can be used for detecting trisomy 21 toreduce the occurrence of false negative result.

The following Table 8 only illustrates the detection and calculationresult of part of samples to be detected, and does not illustrate theresult of other samples.

TABLE 8 Calculation table of Z value of corrected chromosome 21Karyotype Corrected Sample result Corrected X Z value G405 47, XX, +210.014702324 19.36834952 G387 47, XX, +21 0.014591238 17.91101164 G37647, XX, +21 0.014355433 14.81746245 G393 47, XX, +21 0.01404412310.73336561 G491 46, XY 0.013049564 −2.314322329 G641 46, XY 0.0132870670.801496645 G488 46, XY 0.013284892 0.772960653 G408 46, XX 0.013038312−2.461945144 Chromosome 18 Corrected X Average value 0.013225973Standard error 0.0000762249

It can be seen from the above table that, the Z value of samples G405,G387, G376, G393 is more than 3, which can be judged as trisomy 21. TheZ value of other samples is between −3 and +3.

Embodiment 2 A Kit for Detecting the Copy Number of Fetal Chromosomes orTumor Cell Chromosomes

With respect to the detecting method of Embodiment 1, the inventor ofthe application develops a kit for detecting the copy number of fetalchromosomes or tumor cell chromosomes, which includes:

an instrument for collecting blood from a pregnant women or a tumorpatient, which can be any blood collecting needle for collecting blood,syringe or the like;

an instrument for separating blood cells from plasma in blood, which canbe a micro-tube suitable for containing blood on a centrifuge or anyother container or instrument for separation;

Reagents and instrument for extracting DNA from the plasma, which caninclude protease, saturated phenol, chloroform:isoamylol (24:1), sodiumacetate, anhydrous alcohol, 70% ethanol, TE solution etc., the DNA inthe plasma can be extracted by using DNA extraction kit produced byQiagen (product number: 57704) and any other reagents or containers forextracting the DNA;

A reagent and an instrument for preparing the DNA into a sequencinglibrary, the sequencing library can be a library for paired-end shortsequence sequencing, a library for single-end long sequence sequencingor a library for single-end short sequence sequencing, the a reagent andan instrument for preparing the DNA into the library for paired-endshort sequence sequencing includes: T4 DNA ligase buffer with 10 mM ATP,10 mM dNTP Mix, T4 DNA polymerase, Klenow enzyme and T4 PNK (the abovereagents are provided by Illumina sample preparation kit PE-102-1001),as well as ion exchange resin with affinity to the DNA under certaincircumstances to realize the separation of DNA, also, QIAGEN QIAquickPCR product separation kit (product number: #28104) or QIAGEN MinElutePCR product separation kit (product number: #28004) can be selected;

A reagent and an instrument for sequencing the DNA, which can be usedfor performing paired-end short sequence sequencing, single-end longsequence sequencing or single-end short sequence sequencing on the DNA.The a reagent and an instrument for performing paired-end short sequencesequencing can include PE PCR primer PE 2.0, PE PCR primer PE1.0,Phusion DNA polymerase (Finnzymes Oy) (the reagents are provided byIllumina sample preparation kit PE-102-1001).

Embodiment 3 Another Kit for Detecting the Copy Number of FetalChromosomes or Tumor Cell Chromosomes

The inventor of the application develops another kit for detecting thecopy number of fetal chromosome or tumor cell chromosomes, whichincludes:

an instrument for collecting blood from a pregnant women or a tumorpatient, which can be any blood collecting needle for collecting blood,syringe or the like;

an instrument for separating blood cells from plasma in blood, which canbe a micro-tube suitable for containing blood on a centrifuge or anyother container or instrument for separation;

A reagent and an instrument for extracting DNA from the plasma, whichcan include protease, saturated phenol, chloroform:isoamylol (24:1),sodium acetate, anhydrous alcohol, 70% ethanol, TE solution etc., theDNA in the plasma can be extracted by using DNA extraction kit producedby Qiagen (product number: 57704) and any other reagent or container forextracting the DNA;

A reagent and an instrument for separating the DNA with a physicalmethod according to the size of the DNA segments, which can include:agarose powder (Biowest 11860), marker (Takara 100 bp DNA marker,product number: D505A) etc.; and

A reagent and an instrument for sequencing DNA with length with lengthat any point or in any interval within the range of 100 bp to 250 bp,which can include a cutter for cutting agarose gel within a certaininterval.

Preferably, the kit can include a reagent and an instrument foramplifying the DNA recovered from cut agarose gel and preparing it intoa sequencing library.

Embodiment 4 A Device for Detecting the Copy Number of Fetal Chromosomeor Tumor Cell Chromosomes

A device for detecting the copy number of fetal chromosomes or tumorcell chromosomes includes:

a detecting module, which is used for sequencing DNA in a sample ofmaternal plasma or plasma of tumor patient, wherein the sequencingincludes a step of preparing all DNA in the sample of maternal plasma orplasma of tumor patient into a sequencing library to sequence the DNA inthe maternal plasma sample, the detecting module can includes cBotinstrument of Illumina and Genome Analyzer or HiSeq2000 sequencer ofIllumina or SOLiD sequencer of ABI;

a comparison module, which is used for comparing a sequencing result ofthe DNA with a genomic sequence map to determine which chromosome eachDNA sequence comes from and the sequence length of each DNA segment, thecomparison module can be human genome standard sequence database hg19;

a calculating module, which is used for calculating the ratio of thenumber of DNA segments from the chromosomes to be detected to all DNAsegments in the same sample, correcting the ratio according to a GCcontent of the DNA segments from the chromosomes to be detected, andcalculating the variation of the corrected ratio of the DNA segmentsfrom the chromosomes to be detected in a sample to be detected, anddetermining the copy number of the chromosomes to be detected accordingto degree of variation; and

an output module, which is used for outputting the copy number of thechromosomes to be detected.

Optionally, the detecting module can detect all DNA segments in thesample, can also only detect all DNA with length at any point or in anyinterval within the range of 100 bp to 250 bp such as 150 bp to 175 bp,the detecting module can be a module or device for performing agarosegel electrophoresis by including a reagent and an instrument forseparating the DNA in the maternal plasma according to the DNA segmentsize upstream of the detecting device.

Obviously, those skilled in the art should understand that some modulesor some steps of the invention can be implemented by general computingdevices. The modules or steps can be focused on a single computingdevice, or distributed on the network composed of multiple computingdevices. Optionally, The modules or steps can be implemented bycomputing device executable program code, thereby storing them in astorage device and executing by the computing device, or implementingthe modules or steps by making them into each integrated circuit modulerespectively, or making many of the modules or steps into singleintegrated circuit module. In such a way, the invention is not limitedto the combination of any particular hardware and software.

The above is only the preferred embodiment of the invention and notintended to limit the scope of protection of the invention. For thoseskilled in the art, various variations and changes can be made to theinvention. Any modifications, equivalent replacements, improvements andthe like within the spirit and principle of the invention shall fallwithin the scope of protection of the invention.

REFERENCES

-   1. Cunningham F, et al. (2002) In Williams Obstretrics (McGraw-Hill    Professional, New York), p 942.-   2. Wapner R, et al. (2003) First-trimester screening for trisomies    21 and 18. N Engl J Med, 349:1405-1413.-   3. Lo Y M, et al. (1997) Presence of fetal DAN in maternal plasma    and serum. Lancet, 350: 485-487.-   4. Lo Y M, et al. (2007) Plasma placental RNA allelic ratio permits    noninvasive prenatal chromosomal aneuploidy detection. Nat Med, 13:    218-223.-   5. Tong Y K, et al. (2006) Noninvasive prenatal detection of fetal    trisomy 18 by epigenetic allelic ratio analysis in maternal plasma:    Theoretical and empirical considerations. Clin Chem, 52: 2194-2202.-   6. Fan H C, Quake S R. (2007) Detection of aneuploidy with digital    polymerase chain reaction. Anal Chem, 79: 7576-7579.-   7. Lo Y M, et al. (2007) Digital PCR for molecular detection of    fetal chromosomal aneuploidy. Proc Natl Acad Sci USA, 104:    13116-13121.-   8. Bentley D R, et al. (2008) Accurate whole human genome sequencing    using reversible terminator chemistry. Nature, 456: 53-59.-   9. McKernan K J, et al. (2009) Sequence and structure variation in a    human genome uncovered by short-read, massively parallel ligation    sequencing using two-base encoding. Genome Research, 119: 1527-1541.-   10. Harris T D, et al. (2008) Single-molecule DNA sequencing of a    viral genome. Science, 320: 106-109.-   11. Fan H C, et al. (2008) Noninvasive diagnosis of fetal aneuploidy    by sequencing DNA from maternal blood. Proc Natl Acad Sci USA, 105:    16266-16271.-   12. Chiu R W K, et al. (2008) Noninvasive prenatal diagnosis of    fetal chromosomal aneuploidy by massively parallel genomic    sequencing of DNA in maternal plasma. Proc Natl Acad Sci USA, 105:    20458-20463.-   13. Chiu R W K, et al. (2010) Maternal plasma DNA analysis with    massively parallel sequencing by ligation for noninvasive prenatal    diagnosis of trisomy 21. Chin Chem, 56:459-463.-   14. Lewis D E, et al. (2010) Antigenic approach to the detection and    isolation of microparticles associated fetal DNA. PCT, US2010,    #025209.-   15. Fan H C, et al. (2010) Analysis of the size distributions of    fetal and maternal cell-free DNA by double-end sequencing. Clin    Chem, 56: 1279-1286.

1. A kit for detecting the copy number of fetal chromosomes or tumorcell chromosomes, comprising: an instrument for collecting blood from apregnant women or a tumor patient; an instrument for separating plasmafrom blood cells in blood; a reagent and an instrument for extractingDNA from the plasma; a reagent and an instrument for separating the DNAwith a physical method according to the size of the DNA segments; and areagent and an instrument for sequencing DNA with length at any point orin any interval within the range of 100 bp to 250 bp.
 2. The kitaccording to claim 1, wherein the fetal chromosomes or tumor cellchromosomes are the whole chromosomes or partial chromosomes.
 3. The kitaccording to claim 1, further comprising: a reagent and an instrumentfor preparing all DNA into a sequencing library.
 4. The kit according toclaim 2, further comprising: a reagent and an instrument for preparingall DNA into a sequencing library.
 5. The kit according to claim 4,further comprising: a reagent and an instrument for performing PCRamplification on the sequencing library.
 6. The kit according to claim1, wherein the DNA with length at any point or in any interval withinthe range of 100 bp to 250 bp is the 150 bp-170 bp DNA; preferably, theDNA with length at any point or in any interval within the range of 100bp to 250 bp is the 167 bp DNA.
 7. (canceled)
 8. A kit for detecting thecopy number of fetal chromosomes or tumor cell chromosomes, comprising:an instrument for collecting blood from a pregnant women or a tumorpatient; an instrument for separating blood cells from plasma in blood;a reagent and an instrument for extracting DNA from the plasma; areagent and an instrument for preparing the DNA into a sequencinglibrary; and a reagent and an instrument for sequencing the DNA.
 9. Thekit according to claim 8, wherein the fetal chromosomes or tumor cellchromosomes are the whole chromosomes or partial chromosomes. 10.(canceled)
 11. A device for detecting the copy number of fetalchromosomes or tumor cell chromosomes, comprising: a detecting module,which is used for sequencing DNA in a sample of pregnant women plasma orplasma of tumor patient; a comparison module, which is used forcomparing a sequencing result of the DNA with a genomic sequence map todetermine which chromosome each DNA sequence comes from and the lengthof each DNA sequence; a calculating module, which is used forcalculating the ratio of the number of DNA segments from the chromosomesto be detected to the total number of DNA segments in the same sample,and correcting the ratio according to a GC content of the DNA segmentsfrom the chromosomes to be detected; and calculating the variation ofthe corrected ratio of the DNA segments from the chromosomes to bedetected in a sample to be detected, and determining the copy number ofthe chromosomes to be detected according to degree of variation; and anoutput module, which is used for outputting the copy number of thechromosomes to be detected.
 12. The device according to claim 11,wherein the calculating module corrects the ratios of the chromosomes 2,3, 4, 5, 6, 7, 8, 12, 13, 18 and X according to the following function:the GC contents of the DNA segments from the chromosomes 2, 3, 4, 5, 6,7, 8, 12, 13, 18 and X respectively have linear relationships with theratios of the DNA segments from each chromosome to the total DNAsegments, the linear relationship can be represented by y=ax+b, where yrepresents the GC content of the DNA segment from the chromosome to bedetected, x represents the ratio of the number of the DNA segments fromthe chromosome to be detected to the total DNA, a and b are constants,and a is negative.
 13. The device according to claim 12, wherein thelinear relationship between the GC contents of the DNA segments from thechromosomes 2, 3, 4, 5, 6, 7, 8, 12, 13, 18 and X and the ratios of theDNA segments from each chromosome to the total DNA segments is givenbelow: Chromosome No. Function 2 Y = −1274.5X + 154.48 3 Y = −682.68X +88.11 4 Y = −391.42X + 62.075 5 Y = −772.25X + 88.517 6 Y = −729.61X +84.354 7 Y = −2874.7X + 197.28 8 Y = −1599.4X + 122.84 12 Y = −1936.7X +129.32 13 Y = −827.29X + 67.049 18 Y = −1946.4X + 97.323 X Y =−749.77X + 71.33.


14. The device according to claim 11, wherein the calculating modulecorrects the ratios of the chromosomes 1, 9, 10, 11, 15, 16, 17, 19, 20,21 and 22 according to the following function: the GC contents of theDNA segments from the chromosomes 1, 9, 10, 11, 15, 16, 17, 19, 20, 21and 22 respectively have linear relationships with the ratios of the DNAsegments from each chromosome to the total DNA segments, the linearrelationship can be represented by y=ax+b, where y represents the GCcontent of the DNA segment from the chromosome to be detected, xrepresents the ratio of the number of the DNA segments from thechromosome to be detected to the total DNA, a and b are constants, and ais positive.
 15. The device according to claim 14, wherein the linearrelationship between the GC contents of the DNA segments from thechromosomes 1, 9, 10, 11, 15, 16, 17, 19, 20, 21 and 22 and the ratiosof the DNA segments from each chromosome to the total DNA segments isgiven below: Chromosome No. Function 1 Y = 909.17X − 29.372 9 Y =2211.3X − 45.632 10 Y = 1886.8X − 51.026 11 Y = 1255X − 15.714 15 Y =1775.8X − 10.124 16 Y = 797.17X + 23.883 17 Y = 560.83X + 31.227 19 Y =513.79X + 43.088 20 Y = 1006.7X + 22.818 21 Y = 7298X − 51.083 22 Y =596.24X + 43.346.

16.-19. (canceled)
 20. The device according to claim 11, wherein thecalculating module is used for calculating the ratio of the number ofthe DNA segments from the chromosomes to be detected in all DNA withlength at any point or in any interval within the range of 100 bp to 250bp in the same sample to the total number of all DNA segments withlength at any point or in any interval within the range of 100 bp to 250bp, correcting the ratio according to the GC content of the DNA segmentsfrom the chromosomes to be detected in DNA with length at any point orin any interval within the range of 100 bp to 250 bp, and calculatingthe variation of the corrected ratio of the DNA segments from thechromosomes to be detected in the sample to be detected, and determiningthe copy number of the chromosomes to be detected according to degree ofvariation.
 21. The device according to claim 20, wherein the DNA withlength at any point or in any interval within the range of 100 bp to 250bp in the sample of maternal plasma or plasma of tumor patient areprepared into a sequencing library; preferably, the DNA with length atany point or in any interval within the range of 100 bp to 250 bp is the150 bp-170 bp DNA; more preferably, the DNA with length at any point orin any interval within the range of 100 bp to 250 bp is the 167 bp DNA.22.-24. (canceled)
 25. A method for detecting the copy number of fetalchromosomes or tumor cell chromosomes, comprising the following steps:collecting maternal plasma or plasma of tumor patient; separating theplasma from blood cells in blood; preparing DNA in the plasma into asequencing library; sequencing the DNA sequencing library; comparing asequencing result with a genomic sequence map to determine whichchromosome each DNA sequence comes from and the length of each DNAsequence; and calculating the ratio of the DNA segments from thechromosomes to be detected to the total number of the DNA segments bysequencing and comparison results of DNA, correcting the ratio accordingto a GC content of the DNA segments from the chromosomes to be detected,and calculating the variation of the corrected ratio of the DNA segmentsfrom the chromosomes to be detected in a sample to be detected, anddetermining the copy number of the chromosomes to be detected accordingto degree of variation.
 26. The method according to claim 25, furthercomprising correcting the ratios of the chromosomes 2, 3, 4, 5, 6, 7, 8,12, 13, 18 and X according to the following function: the GC contents ofthe DNA segments from the chromosomes 2, 3, 4, 5, 6, 7, 8, 12, 13, 18and X respectively have linear relationships with the ratios of the DNAsegments from each chromosome to the total DNA segments, the linearrelationship can be represented by y=ax+b, where y represents the GCcontent of the DNA segment from the chromosome to be detected, xrepresents the ratio of the number of the DNA segments from thechromosome to be detected to the total DNA, a and b are constants, and ais negative; preferably, the linear relationship between the GC contentsof the DNA segments from the chromosomes 2, 3, 4, 5, 6, 7, 8, 12, 13, 18and X and the ratios of the DNA segments from each chromosome to thetotal DNA segments is as shown in claim
 13. 27. (canceled)
 28. Themethod according to claim 25, further comprising correcting the ratiosof the chromosomes 1, 9, 10, 11, 15, 16, 17, 19, 20, 21 and 22 accordingto the following function: the GC contents of the DNA segments from thechromosomes 1, 9, 10, 11, 15, 16, 17, 19, 20, 21 and 22 respectivelyhave linear relationships with the ratios of the DNA segments from eachchromosome to the total DNA segments, the linear relationship can berepresented by y=ax+b, where y represents the GC content of the DNAsegment from the chromosome to be detected, x represents the ratio ofthe number of the DNA segments from the chromosome to be detected to thetotal DNA, a and b are constants, and a is positive.
 29. The methodaccording to claim 28, wherein the linear relationship between the GCcontents of the DNA segments from the chromosomes 1, 9, 10, 11, 15, 16,17, 19, 20, 21 and 22 and the ratios of the DNA segments from eachchromosome to the total DNA segments is as shown in claim
 15. 30. Themethod according to claim 25, comprising calculating the ratio of thenumber of the DNA segments from the chromosomes to be detected in allDNA with length at any point or in any interval within the range of 100bp to 250 bp in the same sample to the total number of all DNA segmentswith length at any point or in any interval within the range of 100 bpto 250 bp only by sequencing and comparison results of all DNA withlength at any point or in any interval within the range of 100 bp to 250bp, correcting the ratio according to the GC content of the DNA segmentsfrom the chromosomes to be detected in all DNA with length at any pointor in any interval within the range of 100 bp to 250 bp, and calculatingthe variation of the corrected ratio of the DNA segments from thechromosomes to be detected in the sample to be detected, and determiningthe copy number of the chromosomes to be detected according to degree ofvariation; preferably, the DNA with length at any point or in anyinterval within the range of 100 bp to 250 bp is the 150 bp-170 bp DNA;more preferably, the DNA with length at any point or in any intervalwithin the range of 100 bp to 250 bp is the 167 bp DNA. 31.-33.(canceled)